2.1 Biology and Life Cycle of Coccidial Protozoa
Coccidial protozoa may infect and inhabit the gastrointestinal tract of most species of warm-blooded animals, most often and most seriously when the animals are penned or in concentrated groups. For the most part infection occurs with coccidial species specific to the animal host species and more than one coccidial species may infect a given host animal species. For example, seven major coccidial species are known to infect the chicken.
The life cycle of the coccidial parasite involves several complex stages. Under natural conditions the cycle begins with oral ingestion by the host animal of excrement from another animal of the same species having sporulated oocysts dispersed therein. The sporulated oocyst shell then breaks down by action of the digestive system of the host animal. For example when the host animal is a chicken the outer shell of the sporulated oocyst is broken down mechanically to liberate first the sporocysts and finally biochemically to liberate sporozoites which are the ultimate infective organism. Other stages in the cycle which follow redevelopment within about 7 days include schizont I, merozoite I, schizont II, merozoite II, gametocyte, gamete and completion of the cycle with oocyst formation.
More descriptively, in the instance of the chicken, natural infection with one of the important species of Eimeria, i.e., Eimeria tenella begins with ingestion of an overwhelming amount of the sporulated oocysts. Each sporulated E. tenella oocyst contains four sporocysts and each sporocyst contains two sporozoites. In the stomach, the sporulated oocysts rupture, freeing the sporocysts. The sporozoites are liberated from the sporocysts by enzymatic action and pass down the digestive tract. Each of the sporozoites eventually reach the cecal pouches where they enter epithelial cells of the cecal mucosa and start an asexual multiplication cycle. The details of the completion of the life cycle referred to above are well known in the art.
The rationale of vaccination with live sporozoites, in the present invention, is to set up a low level of infection (subclinical) during the first few days of life of the animal, e.g., a chicken, without slowing down the performance of the animal in terms of growth. The subclinical infection, thus established induces immunity, while the host, e.g., chicken, has the advantages of passive protection afforded by maternal antibodies. The maternal antibodies also help to down regulate infection, thus helping to decrease unnecessary parasite buildup in animal houses, which is critical for the success of a vaccine since the degree of severity of disease is directly proportional to the numbers of sporulated oocysts ingested by the host from its environment. Under field conditions chickens, e.g., have their maternal anitbodies reduced to very low levels by the third week and hence become susceptible to the parasite, if their immune system is not primed by vaccination. Without vaccination the challenge encountered by hosts is frequently devastating, especially to concentrated populations of animals resulting in death or illness and/or poor development or growth quality and subsequent economic loss to the animal grower.
2.2 Prior Related Vaccines
U.K. Patent Specification No. 2,008,404 describes a method of promoting the growth of poultry by feeding viable sporulated oocysts of coccidia to poultry. Similarly chickens have been said to be immunized against cecal coccidiosis with viable sporulated oocysts in the disclosure of U.S. Pat. No. 3,147,186.
An immunogenic composition for oral administration comprising live encysted protozoa such as coccidia is described in British Application No. 2,144,331A wherein encysted sporulated coccidial oocysts or encysted coccidial sporocysts embedded in a firm gel matrix were fed orally to the host animal such as a newly-hatched poultry chick. Gels based on alginate were used for the matrix.
Sharma, N.N. in The Journal of Parasitology Vol. 50, No. 4 (1984) pp. 509-517 describes unsuccessful immunization trails with sporulated viable oocysts of coccidia or with an impure mixture said to contain sporozoites along with oocysts and sporocysts of coccidia administered to chickens, orally, intravenously, intraperitoneally, intramuscularly or subcutaneously.
2.3 Problems Solved
Since the severity of disease is directly proportional to the number of sporulated oocysts in the field available for ingestion, existing vaccines based on either feeding sporulated oocyst or feeding microencapsulated oocyst have limited utility for the broiler chicken industry, in that the high numbers of oocysts available for ingestion from day one often causes severe infections. As a result, the use of oral vaccines as such often necessitates the use of anticoccidials. Therefore the use of sporulated oocysts as live vaccines have limited application. Additionally, sporozoites are ineffective in producing an infection when administered orally to chickens.
Heretofore unsuccessful attempts to create immunity with unprotected sporozoites by systemic innoculation have been made by Sharma, supra., where attempts to create infection with sporozoites have been confined to use of freshly excysted sporozoites inasmuch as methods have not previously been available to keep the sporozoites alive longer than a few days under refrigeration. Consequently vaccination using sporozoites was impractical commercially.
The preferred method of this invention of microencapsulating by finely dividing a suspension and hardening the water-soluble polymeric stabilizer to enclose a physiologically balanced suspension of sporozoites provides time flexibility as to when the vaccine must be used, since the lifetime (in terms of viability and infectivity) of the sporozoites is extended up to at least 5 weeks. The second method of this invention of using water-soluble polymeric stabilizer, preferably a water-soluble alginate salt to suspend sporozoites in a medium physiologically balanced with respect to isotonicity and pH has extended the lifetime of the sporozoites up to at least 2 weeks and thus also provides time flexibility as to when the vaccine must be used.
The technique of microencapsulation of sporozoites used here not only provides a practical way of vaccination of chickens when they are handled on their first day of life, but also offers other advantages, such as, e.g., avoiding buildup of oocysts in the animal yard or pen which occurs both during vaccination as well as afterwards due to parasite multiplication in the host when live oocysts are used commercially, e.g., spread in the yard or pen or used in feed to innoculate, and thus avoids early overwhelming challenge due to that source of oocysts. Use of alginate suspension and microcapsules containing suspensions of sporozoites, in contrast, results in much slower and lower levels of oocyst buildup underfoot.